国立公害研究所研究儲告 第18号

RとsearchReportErom TheNationaトInstituleEor EnYironmentaJS－udies No．18．1980

R－18－’80

α≠fi鋤αfわ几

ぶぬ花dαrd虎e花reんce肋わrねJ

Ⅰ姐ited by Kensaku OKAMOTO

THE NATIONALJNSTITUTE FOR ENVZRONMENTAL STUDIES

環境庁 国立公害研究所

■ ・．■■．■＝－－－－・－・・・l

＝

、－1－－－‖－卜l－．

、し≠丑」り〃り．し

Preparation，Analysis and Certification

of PEPPERBUSH

Standard Reference Material

Edited by

KensahlOKAMOTO

DivisionofChemistryandPhysics

TheNatlOnalInstituteforEnvironmentalStudies

PEやPER8USH PLANT

rC王e比和あ8rあ血e和白翫eb．如Zu¢e．ノ

Prゆce

MeasurementofthephysicalandchemicalpIOpertiesofsubstancesisfbndamentaltothe 鮎1d ofMaterialscience．Inordertounderstandthechemicalpropertiesandbehaviourof material，knowledgeofelementalcomposition享ntermsofaccuracyandprecisionisrequired・ Withthelatterobjectiveinmind，Standardreferencematerialsofdiversecategorieshave beenpreparedandissuedbyinstitutionsinanumberofcountriesformanyyears・

WhenoufinstltuteWaSinauguratedin1974，thepreparationofenvironmentalstandard referencematerialswaschosenasoneofthelong．temlreSearChprqjectsfortheDivisionof ChemistryandPhysics．Itismygreatpleasure，therefore，tOWritethlSintroductorynoteon “Pepperbush”，thefirststandardreferencematerlal，tObeissuedbytheinstitute・

Dr．K．Okamoto，Ofthe BiochemicalAnalysis seetion，WaS Chiefinvestigator ofthe prq）eCt tOgeth9rwithDr．Y．Ambe，head ofthe AirQualityandBiochemicalAnalysis SeCtions・Dr・C・McLeod，visiting reseaICher from山e United Kindgom，undertook［CP analysisofPepperbushwhileDT．T．Takamatsu，amemberoftheDivisionofWaterandSoil Environrnent，PrOVidedresultsbyX・rayfluorescencespectrometry．Dr．Murozumi，Professor ofMuroranInstitute ofTechnology，gaVe aSPeCialcontributioninprovidlngreSultsby isotopedilut10nmaSSSPeCtrometry・TheisotopedilutlOnteChniqueisindispensableforthlS kindofworkandwearemostgratefu1fortheeffortsofDr・Murozumi・AspecialtharLksis alsoduetoProfessorT．KibaandhlSreSearChgroupforincludingPepperbushasoneoftheir Samplematerialswithinthespecialenvironmentalresearchprogrammewhichwassupported bytheMinistryofEducatlOn．

鮎ptember，1粥0．

KeiichiroFuwa

Chlef，Divisionof

Chemistry＆Physics．

CONTENTS

page

Preface

Chapter I StandardReferenceMaterialsin Environmental Analysis

NatureofPepperbushPlant

PreparationofPepperbush

AnalysisofPepperbushhy AtomicAh50rPtlOnandFlame Emission Spectrometry

ElementalAnalysisofPepperbush hyICPEmis＄ionSpectrometry

Y．Ambe…………・・ 1

K．Okamoto……・… 7

K．Okamoto………・20

Chapter II

Chapter III

Chapter IV

K．Okamoto………25

Chapter V C．W．McLeod，‥＝…33

N．Furuta，

M．Nishikawa

AnalysisofPepperbushby Energy一血spersiveXィay

Fluorescence Spectrometry

DeterminationofAlkalies， AlkalineEarthsandSomeHeavy MetalsinPepperbu5hbyIsotope DilutionMassSpectrometry

Cllapter VI

T．Takamatsu…・・・・54

ChapterVII

M．Muro乙uml，・＝‥‥60

S．Nakamura，

T・Igarasl厄，

K・Suga，

K．Yoshida

K．Okamoto………77 Chapter VIII TheCertificationofPepperbush

Acknowledgements

K．Okamoto・‥・…‥ 95 SummaryinJapanese

⊥fJr O／C加〝fわuJ〝J

yo∫鋸〃α〟A〟βg

AirQualityMeasurementSectlOn，Chemistry andPhysicsDivision，NIES・

人川∫dhJ（）〟．1。IJO几）

BiochemicalAnalysis Section，Chemistry and Physics Division，NIES．

α椚e／0〃W．〟正月クβ

BiochemicalAnalysisSection，Chemistry andPhysicsDivision，NIES，and DepartmentofChemistry，FacultyofScience，UniversityofTokyo・

〟〃0加F【ノRm

WaterQualityMeasurementSection，ChemistryandPhysICSDivision，NIES・

肋∫αJ（止α〟搭仕肱4帆4

LaboratoTy ServicesandMonitoringSection，Chemistry andPhysicsDivi－ Sion，NIES．

乃桓軌けM∽肱灯駅 SoilEnvironmentalSection，WaterandSoilEnviTOnment Division，NIES．

肋5町0〟〔偶OZ【瑚ぶeヴ川1朋〟こ胤4．乃Jmざ鋸JGA且AぷⅣ，片αZ〃γα∫【／GA

＆肋f双J∽fyO∫J〃かA．

Department of Applied Chemistry，Muroranlnstitute ofTechnology・ Mizumotocho27－1，MuroranshiO50，Hokkaido．

V

CHAIyrERI

STANDARD REFERENCE MATERIALSIN

ENVIRONMENTAL ANAl」YSIS

Y．Ambe

♪汀rod〟CJわ〃

hrecentyears，anincreasingnurnberofchemicalanalysesofvarlOuSklndsofenviron－ mentalsamples，SuCh asthe atmosphere，Water，SOilandbiologicalmaterials，havebeen carriedoutinlaboratoriesofuniversities，nationalandlocalgoveTrmentalorganiヱatlOnS，and independentinstitutions・ThemainpurposeofsuchanalysesaretoindlCatetheactualstate ofenvlrOnmentalpo11ution，detecttheonsetofadversechangesintheenvironmentandto understanddle Origlnandmechanismsofthephenomena・Thepollutantstakenasobjects forenvironmentalrnomitoringincludenotonlythosecontIOuedlegal1yastoxicsubstances butalsosuchchemicalsthatarepotentlalhazards．Inordertocharactedzethedistribution ofpollutantsintheenvironment，and／ortodetectabnormalvaluesinatimeseriesovera longperiod，alargenumberandawidevarletyOfenvironmentalsamplesareco11ectedand andyzed・Unfortunatelythe reliabilityofdataprovidedbylaboratoriesengagedinsuch prqectsisopentoquestionsincethereisalacknotonlyofappropriatestandardIeference materials butalso reference methods ofanalysis．lnmanyinstance＄，therefore，itis not

possibletoproperlyassesstheaJlalyticaldataandtodecidewhetherthedataconstitutesan environmentalproblem・ItismainlyforthisreasonthattheNatlOnalhstituteforEnviron・ mentalStudies（NIES）has recentlyinstigated a standard reference materlarS（SRM） progfamme・Thisintroductorychapterdescribesdle ChemicalcharacteristlCSOfenviron・ mentalsamplesanddlSCuSSeStheconceptoftheSRMwithinthe丘ameworkofenviron・ mentalanalysis・ThestatusoftheSRMprogrammeatNIESisalsosummarlZed・

仇踊腑血血げ助血洲W血愉明血

First，WeShouldconsiderthecharacteristicpropertiesofenvironmentalsamplesfrornthe vleWpOintofchemicalanalysIS・＝ere，environmentalsamplesincludeair・Water・SOil・plant andanimaltissue，COntainingornotcontmiiTlgPOllutantsdlreCtlyhazardoustoourhealth， andincludethosemoughtofasindicatorsformonitoringenvironmentalpdLl11tion・

1）MulticompoTlentSyStemS

Environmentalsamplesarecomposedofmanyelementsandcompounds・andsometimes differentphasesco－eXist・Thecomplexcompositionofthematerialsmayinl1ibitaccurate analysisoftheobjectlVeeOmpOnentS．Forexample，theco－eXIStenCeOfmaJOrelementsmay sometimescausematrixinterference，increasingordecreasingthevalueundermeasurement・ Alsothechemicalcompositionofenvironmentalsamplesdiffergreatlysothattheappro－

ー1－

prlate analyticalmethod dlOuldbe selectedconsideringthe matrix ofthe analyte．Very OftentheanalyticalpTOCedurerequlreSaChemicalscpaIat10nOfdleCOmpOnentOfinterest たom仇eor噛n山mate血．

2）HeterogeneouscompositionaJlddistributionofpollutaJltS

Biologicaland geolo由Calsamples，Characteristical1y，areheterogeneousin composition and pollutants may be distrib11ted heterogeneously accordlng tOtheir physico．chemical propertleS．Inanlmalbody，halogenatedpesticideslikeBHChaveatendencytoconcentrate inthe fatty part ofthebody，and，therefore，COnCentrationsdlfferaccordingtothesites analysed．Itisalsoin1pOrtanttOSpeCifywhedlerleSultsareexpressedona“total”oT”fat” basis．Inthiscase，the umitstoexpresstheconcentrationsdlOuldbecarefu11yselectedto PrOVidethemostmeaningLhlresults．

Whenweintendtoexpressconcentrationsonatotalbasis，COmpletehomog印atlOnOfthe Sampleisnecessarypr10rtOa叫ysis・ItisradlerdlmCult，however，tOPreParehomogenous SamplesforsometypesofmaterialsuchashumanhairoI丘sh．

3）Wideramgeofcomcemtratioms

Concentrationsofpollutantsmayvaryfrompptlevelstosevefalhundredsofppmsothat theselectionofanappropriateanalyticalmethodfortheconcentrationlevelexpectedisan essentialpoi山．

Forthe detemlination oflowlevelconcentratlOnS，thehandlingandpretreatmentof Samplemustbe carefu11y performed takingcaIetOpreVentCOntaminationorloss．Special fac山tiessuchasacleanlaboratoryareofYaluetominimizecontaminationfromalrParticu－ 1ates．Referencemethodsofanalysisfordeterminationsattheppborpptlevelarerequired and，inspiteofmanyefforts，fewsuchmethodshaYebeenestablished．

4）Complexchemicalfomsofcompomemts

Itiswellknownthattheessentialityandtoxicityofaelementarecloselyrelatedtonot Onlythetotalamountofametalbutalsothechemicalfbrmsoftheelement．Animportant requlrement，dlerefore，isthatanalysisinvoIvingchemicalspeciationshouldbeperformedto evaluatethequalityofourenvironmentcorfeCtly．

ThechemicalfomlS Ofanelementmay beexceedinglycomplex，andtheexistenceof yadousoxjda出on5ta旭，肌dche血cdb加血相暮0】owmolecu】arWeiかCOmpOund55UCh且Sa metallo・OrganlcsubstaJICeand／orhighmolecularYeightcompoundssuchasametalloprotein Shouldbeconsidered．Theidentincationanddeterminationofeachchemicalspeciesinvari－ ablyrequlreSaPrellminaJySeparationofthecomponentofinterestpr10rtOdetemination．

The detemlination oftotalamounts ofelementshasbeenextensively cardedout for enYironmentalstudies，butitismuchmore dimcult to determinethe chemicalfomlSOf

elements．The developmentandimprovementofseparation techniqueswhichcauselittle denaturationoftheLnvivocomponentsarerequired・

ThissituationisalsotrueOforganiccompoundswhichhavemanyderiYatives．

5）Stab払ityo一組mples

During tranSpOrtation ofthe sample fromthe collectlOn pOint tothelaboratory substantialchangesintheamountsandchemicalfomlSOfpo11utantsandsamplemediamay occur．care mustbetakentoselectthe appropriatesampler，COntainerandpreservatlOn procedure・Generallyenvironmentalsamplescanreadilyundergophysiolodcalandchemical

chaLlgeSduetomicrobialactivity・Thercfore，themostsuitablepreservationmethodssuchas

ー2－

acidi鮎ation，freezlngOrSterilizationshouldbeappuedtothesample・ AshlentlOned above，many featuresofenYironmentalsamplesmake accurateanalysis

difficult and the proper selection oftheanalytlCalmedlOdsandtechnlqueSisrequired・ Analyticalmethods血ouldbeselectedconsideringnotonlythenatureofthesamplebut alsosensitlVityandinl1ererLtaCCuraCyOfthe analytlCaltechnique・Thefacilityandinstru・ mentsoflaboratoryandtheexperlenCeOfascientistarealsofactorswhichdetemlinethe analyticalmethodsandtechniquestobeused．

仇eo／∫ね〃（お′d月e陶′e〃Ce肋Je血JJ

The various application areas ofSRMsin measurement systemshavebeen discussed extensIVelyinaNBSbookletl）andarebrie爪ysummarizedbelow： （1）TheSRMcanbeusedasastandardtocalibrateameasurementsystem．Namely，by

analysingaSRMusingaspecifiedmethodortechnique，theaccu－aCyOftheanalytical medlOdcanbechecked．

（2）Indevelopinganewanalyticalmethodtheaccuracy且ndprecisionofthernethodhave tobejudged．SRMscan，therefore，SerVetOeStabli血thereliabilityofthenewmethod・

（3）The SRMcan beusedtoassurelong－temqualitycontrolparticuladyforroutineand 鮎1dappucationsbyanalyslngtheSRMatappropriateandregulartimeintervals・Inthis case，thepreservationoftheSRMshouldbeguaranteed，Inthe鮎IdoferlVironmental 弧旭n00，甲山吋COntTOlforgovernmentalandloc血1e由或ationisofvit止血poftanCe、As mentionedearlielalargenumbefOfdataonawidevarletyOfenYironmentalsamples havebeenreported frommanylaboratories，butthesedata somctmeslackanassur－ anceofaccuracysincedatahavebeenobtalnedbydifferentanalystsofdiverseexperi－ ence andby diffeTentanalyticalmethods．Thus，thevalue oftheSRMirLthlSareais Cleadyreco騨ized．

舶画血頼再臨血刷叫加附加血融

nefollowingitemsaTeCOnSideTedtobeIe中1isitesfo一也eSRM－

（1）Certi爪edmater餌

Itis a fundamentalpointthat theanalyticalYaluesofthe components areweu・ characterized and certl鮎d．Such numerlCalvalues are temled”certi鮎d values”and areconsideredtobethemostaccuratewithlnthelimitationsimposedbyequlPment andmethodology・AdequatcinformatlOnOn thecertifiedvalues，dleunCertaintiesof thevaluesandinstrucdonsonsample preparationshouldbespecined・Detai1softhe CertincationprocessforPepperbusharegiYeninChapterVIJI・

¢）Demandft〉Tmatedd SRMswereorignia11ymadeinfieldsofindustryandcommerce・forexample・tOaSSist inmeasurementandexchangeofgoodsl）．lnrecentyearsseveralcategoriesofenYiron－ mentalSRMshave beenprepaIedinordertoimprovethe qualityofenvironmental analysis．1tisnotunreasonable，therefore，thatbiologicalarLdenvironmentalSRMs havebeenissuedbytheNationalBuIeauOfStandards，U・S・A・PBS）andothergovcm－ mentalorgarLIZationssucethe1970，stomeetthegrowingdemandsofenvironmental scientists．

（3）Homogeneous弧dstablemate血1 Thehomogeneityofcompositionandstabilityofeachaliquotorbotdeofsample

－3－

must be guaraJlteed．By de餌Iition the SRMis ahomogeneousandstable materlal． Liquid orpowderedsamplesare favorable forthe preparationofa homogeneous material．In prepanng a SRM，preCipitates orlarge particles which causeinl10mO－ geneityofthematerialshouldbe removedby filtrationorsleVing・Jtisalsoessential thatthestruCture，COmPOSitionandpropertiesoftheSRMwi11notchangeoYeralong Period sothatthe certinedvalues，OnCe detemined，Wu bevalid fbrmorethanten years・Varioustreatmentsorstoragemethodssuchassterilizationby －rayradiation， germieide addition，Or COld storage，etC・Shouldbeutilizedforlong－termStOrage Of SRMsoflowpreservability・ContalnerSforSRMsalsoshouldbecarefh11ychosenso thatchangesofcontentbycontaminationoradsorptionlossdonotoccur．

（4）SRMcomposition Itispref如ablethatthecompositionoftheSRMshouldbeclosetothatofthesample beingarlalysed，0therwiseitmaynotbepossibletomakejudgementsonthereliability OfanalysIS・The potentialerrors on trace element determinationscaused bymqjoT matrixcomponentsisoneaTeaWhichshouldbeconsideredwhenassesslngdata・

（5）bng・tem飢Ipply

AsumcientquantityortheSRMshouldbepreparedtosupplyusers’needsforatleast 5・10years・SpeedydlStributionthTOughanappropriateorganizationisalsorequired forend・uSerStOreCeiveSRMsassoonaspossible．

¢）Av姐ab山野at10WCOStalldsa食中伽h弧甲Or也tion

〟如orお〟J助cたgm〃〃如〃d伽Je〝J∫ね血相（げ

g郡わ〃me〃JαJぷ尺〟丹0ダ伊乃meαJ〟郡

SRMswere鮎stpreparedin1904byNBS；themetalsamplesbeingissuedinchipfom． Muchlater，irL1955，theU．S．Geolo由CalSurvey（USGS）preparedstandardrocksamples， G－1andW－1，WhichweredistributedtoaJlalyticallaboratoriesthroughouttheworld．Inthe 1960’s some studies concemed with“soft’’standardswere perfc，rmed，in addltion to COntinuedis＄ueOf“hard”sRMssuchassteels，由ass，andcement，etC，Bowen’sstudyonKale POWderbeganin1960andinthisinvestigation，42elementsweredeterminedatmany laboratories usinglldifferent analyticalmethods2），The metalcontentofabone－aSh standardwasstudledbyVallee3）．since1970aseriesofbiologicalandenvironmentalSRMs have beenissued by NBS・ne NationalBuTeau OrStandards SRM categoriesinclude “OrchardLeaves”，■“BovmeLiver’’，“TomatoLeaves”，“Spinach”，”PineNeedles”，‘‘Oyster Tissue”，“RiverSedlment”，“AlrParticulateMatters”，“Trace ElementsinWater”andso

forth・TheIntematlOnalAtomicEllergyAgencyhasalsodistributedsomecertinedrefeTenCe materials or－eference mat¢血1s such as“So封’’，“bke Sedim即が’，“加ed Cop叩Oda”， L’HomogenizedFishFlesh”andetc．

InJapan，0nly twokindsorstandardsamplehave beenutilizedinthe environmental

science neld，Olleisaseawatersampleforsalinitymeasurementandthestandardiscalib・

ratedversustheInternationalStandard SeaWaterpreparedbytheInstitute ofOceano・ graphicSciencesatWorrrdey，England，Whnetheotherisstandardrocksamples，JB－1，JG・l preparedbydleGeologicalSurveyofJapan・Researchmaterlalssuchastealeavei，Shark rneat，bamboo，andfungalmycelia，haverecentlybeenstudiedascandidatesfoTSRMs4，5）・

NIEShasrecendyinstigatedaSRMprogramme，theobjectivebeingthepreparationand ceT11ficationofenvi10nmentaistanda通，tOSelVetheneedsofenvuonmentalscientistsand

laboratorleS．Vario11SCategOriesofSRMscanbeconsidered，forexample・elements，gaSeS，

一4－

Organic compounds・At present，OuISRMpIOgrammeisrestricted tothe pIeParation of

environment山samplesandcerti丘cationofelementalcompositlQn．

Tablel

NIES SRMs

Pepperbush

Pond Sediment

Chlorella

Human Serum

Halr

（Bota山c叫

（Geolo辞Cd）

（BiologlCal）

（Clinical）

（Clinical）

Cer也丘ed

Analysisinprogress AnalysisinprogIeSS Analysisinprogress Analysis in progress

TablelshowsdlCpreSentStatuSOftheSRMprograTrmeatNIES・ThefirstSRMtobe completedisabotarLicdsample，Pepperbush，thepreparatlOn，analysisandcertificationof whicharedescribedinthisbook・ReportsontheremainingSRMswi11alsobeissuedindue course．ThemosttypicalcharacteristicsofPepperbushrelativetothebotanlCalSRMsissued byNBSarethehighconcentrationsofmanganeSe，Zinc，CObalt，nickelandcadmium・

The seeondissueisageolo如alsample，PoJldSed血ent，thecertincationofwhichis presendybeingperformed・SanshlrOpOndwithinthegroundsoftheUniversityofTokyo， wasselectedasthesiteforcollectlngthesedlment・InMay1977，Whenthepondwasbeing dredgeddurlngaCleaningoperation，1argeamountsofsalvagedsedlment（about500kg，Wet wt．）weretakenandtransportedto山einstitute，Afterremovalofcoarsemate血11argerthan 2mm，thesedlmentSamplewascolleetedonⅢterpaperandwasair－driedforabouttwo weeks．The procedurea鮎rthisstepwasalmostthesame asthat foTPepperbushSRM・ exceptforthemeshsizesusedinsieving．Thesedimentsampleshavingbeenpasscdthrougha 200．mesh（71pm）nylonsievewerehomogenizedinarlf11esampleramdthenpackagedin 2000glassbottles・（20geach）・Thesamplesinpackagedformweresterilizedby60co radlationat2Mega－RadattheJapaJIAtomicEnergyResearchInstitute（Takasaki）・The Yariationofmetalcontentamongbottles（estimatedbyAASandXRFdeterminations）was negli由blysmal1，indicatingthatthepreparedporLdsedimentsamplesarehomogeneousand practicallyusehdasaSRM・Also，thecomparisonofmetalcontentsbetweenPondSediment andPalaceMoatSediment（Tokyo）6）showsthatthecompositionsaresimdarand，therefore， PondSedimentSRMmaybeconsideredasatypicalsedimentsample・

ThethirdSRMisabiolodcalsample，ChloTella，thepreparationofwhichhasjustbeen completed・ChloreuaisatypicalgreenalgaewhichiswidelydlStributedinlakes・riversand ponds．Inrecentyears，enVironmentalpouutionoftheaquasystembyheavymetalsand organiccompoundsishavingadverseeffectsorlanimals，Plants，tish，and血soonhuman health・Ånurgentrequlrement，therefore，istoidentifyandmonitorsuchsubstancesinthe hydrosphere・BesidesitsvalueformonltOringwaterpollution，Chlore11aSRMwi11alsobeof useinbasicstudleSSincechlorellahasalreadybeenusedextensiYelyasaresearchmaterial・

Itis we11knownthatcertaininorganicelementsarerequiredfornormalgrowthand developmentofthehumanbodyand，atpreSent，about26elementsareconsideredtobe essentialor benencialforlife．Diseases have been related to abnormalconcentrat10nS （dencienciesandexcesses）ofmaJOr，minorandtraceelementsinnuidortissue・Itisclear thatthereisagreatneedforhumanfluidandtissueSRMsand，therefore，ahumanblood serumSRMhasbeenpreparedandanalysisisirlprOgreSS・Humanhairisalsoanimportant

－5一

SamPle for monitorlngm¢talaccumulationin dle human body and such a standardis presently be血Ig prePared．1t should be realizedthat concentrationslevels for many essential／nonLeSSentialelementsinclinicalsamplesareextremelylowandreliablemethodsof analysISarenotaSyeteStablished，andthustheceEtificationofclinicalSRMsisbynomeans an¢aSytaSk，

Asmentionedearlier，theSRMprogrammeatNIEShasbeenorientedtothepreparation Ofdiverseenvironmentalsamplesandcertincationofelementalcomposltion・CleaIlythereis a paranelneed for SRMs wherethe contents ofvanous organiccompoundshavebeen Certifieq・Although there exist many difnculties with respect to 血e preparation， homogeneity，PreSerVatlOn，andanalysisofthe”organic’’SRM，rCSearChformakingsuch SRMsistobecarriedout．

Refe陀neeS

l．Cali，J．P．etaL，（1975）＝TheroleofstandardreferencematerialsinmeasuIementSyStemS． 」Ⅵi∫〟0〃Ogr岬力148．

2．Bowen，H．J．M．（1967）：A′lゆ519ユ，124． 3．Vallee，B．L∴PerSOnalcommunication． 4．FllWa，K．erαJ．（1978）：仇此C加mJ加．ノ叩α〃51，1078． 5．Dokiya，Y．eJ〃J．（1978）：d仙止（乃僧服50，533．

6．Goldbe柑，E．G．eJαJ．（1976）：Geoc力e椚．エ10，165．

一6－

CHAPTERIl

NATUREOFPEPPERBUSHPLANT

K．Okamoto

血けα九Jぐrわ〃

Pepperbush（C7ethnbarbinervisSieb・etZucc），adeciduoustreeof3－7minheight，is widelydlStributedinforestedreg10nSOfJapan．Fromthebiogeochemicalandbiochemical pointsofview，rt，Pperbu血hastwoanomalouspropertieswhichcharacterizethistreefrom Othernomlalplantspecies．First，theabilityoftheplanttoaccumulatecertainheavymetals intheleaveshasbeenestablishedl－3）．AnalytlCaldataonthemetalcontentsoftheplant andotherspeciesarepresentedinsectionl．Thechemicalformsofthemetalsinpepperbush leavesandthemolecularweightsofthespecieshavealsobeeninvestigatedandaredlSCuSSed insection2．1nparticular，thechemicalformofcopper，andthepurificationandpropertleS Ofacopperprotehisolatedfrompepperbushleaveshavebeenstudledandtheresultsare descdbedinsectlOn3．TheunusualabnityoftheplanttoaccumulatecertainheaYymetals also posesthequestionofwhetherhlghlevelsofcertainmetalsarerequiredfornormal growthofthe plant．TheessentialityaJldtoxicityofcobaltforpepperbushandthehter－ Ce11ularmetalinteractionsbetweencobaltandotherelementshavebeenexaminedandpart Oftheresultsarepresentedbrienyinsection4．

Theaccumulat10nOfheavymetalsinpepPerbushhasnutritionalsignincanccsincefre血 1eavesareedlble2，4）．lnoldentimes，yOungleaveswerestockpiledbyorderofthelawasa PreCautionagalnStfamine．Theplantalsoprovidesasourceoffoodforwildanimals，These factsindicatethat pepperbushleaves can supply cobalt and other essentialtrace metals WhicharerequiredfofnOmlalgrowthanddevelopment．

Further，itiskJlOWnthatpepperbushplantgrOWSwe11inareasofvoIcanlCaCtivityand alsonearore re鮎Ieries，SuggeStingthat pepperbushplantistoleranttoexposuIetOSulfbr COmPOunds．Biochemical research to clarify the mechanismof adaptation has been performedintheEnvironmentalBiologyDivisionoftheinstitute5）．Theresultsindicate that pepperbush plaJlt OCCupies aunique positioninthe plantkingdomwithrespect to envlrOnmentalpollutionbysulphurcompounds．

So farthe botanicalSRMswhichhavebeenissuedarecharacterizedonlyintermsfor elementalcomposition．Thereisaneed，however，rOrkJ10wingnotonlythetotalamountof aparticularelementbutalsothechemicalfbml．Itisanticipatedthattherewillbeademand intheneal餌tureforrangesofSRMswhichprovideinformationonthechemicalformof elemelltS．

－7－

J．ア申pe沌〟成，α〟eJαJゼぐCぴm〃ねね′鞄〃乙

Some plantSwhichselectively accumulateelementssuchascopper，Zincandselemium havebeenextensivelystudied6），butlittleisknownabouttheaccumulationofcobaltby higherplants・CTethTYlbarbine血（pepperbush）wasfoundbyYamagataandMuIakamil）to accumulatecobaltininvestigationsonthevariationintheheavymetalcontentofvegeta－ tion．Morethan one hundredtlmeSaSmuChcobaltandseveraltimesasmuchnickelweIe

detectedintheleavesofCbabinervisasinotherplantspeciesl，2）． Tablel血owstheanalyticaldataondleheavymetalcontentofnvenativeplantspecies

growingatthesamesite3），ThemanganesecontentoftheleavesofCba，bineTVisranged froml190to2600ppm（pg／gdryweight），beingmarkedlyhlgherthanthoseofother plants・ThezinccontentofCbarbb7erVisrangedfrom360to440ppm，WheIeaSthoseof Otherplantswerebetween28aJld72ppm・Thezinccontentoflandplantsgrownonnomlal SOilisseldominexcessoflOOppm，SOthatthevalueforCbarbineTViswasexceptional1y high・C barbine”ishadanextremelyhighcobaltcontent，randngftom8to32ppm．In COntraSt，thoseoftheotherfourplantswerelessthanO・5ppm・Fromthreetoseventimes asmuchnickelwerealsofoundinCbarbinerpiscomparedtootherspecies・Itisnoteworthy thatC barbinerviscontalnedextremelyhlgh1evelsofcadmium，rangingfrom6．6to8．O ppm・hthesamplesco11ected丘omGinzandaira，C barbLnerviscontainedbetween about thirtyandeightytimesa軍muChcadmiumasQ，SerrataandQ．mongoticavar．grosseselrata， respectively．

Onthe otherhand，theamountsofiron，COpPerandleadinC bwbinerviswerenot SigllEicantlydlffbrentfromthoseoftheotherfourspecies，aSShowninTableI．Thedataon the heavy metalcontentin various plant speciesshowsthatC barbinervisaccumulates manganese，Zinc，CObalt，nickelandcadmlumintheleaves，Whereasthereisnoparticular amnltyforiron，COpperaJldlead．

2．αemfcαJ凡〉mJO／5b∽e〟eね加ゎ乃押erム〟ざわ飽〃r

ltisofinteresttostudythechemicalformsofthemetalsaccumulatedbypeppeIbush pl且ntbuta5yetl加eisbowれaboutめeざ11坤Ct．50ねーぬepresenceora両肌1in812－1ike cobalt－POrphyrincomplexhasbeenindicatedbybioassayusingmicrobes2），Thepu亜cation Ofvariousmetal－bindcompounds丘ompepperbushleaves，therefoTe，hasbeenperformedin Ourlaboratoryoverthepastfewyears．

1nordertoexaminethechemcialformsofmetalsinpepperbush，thebufferextractfrom

pepp即bu血1eaveswasappliedtoagelc九romatographicco】umlli几Ordertoes止mateappro・

Ximate molecular weights ofmetalcompounds，Fresh pepperbushleaves（250g）were groundwith50mM phosphatebuffer（pH6．8）in amechanlCalmortarfor30min．The

SlurTyWaSCentrihlgedat18，000xgfor20minandthenthesupematantwaslyophilized▲ ThelyophilizedpowderwasdissoIved（100mg／nl）inlOmMMgC12andthenlrnlofthe 5amp】egolutjonwasapp】iedtoa8jo・Ge】PJcdumn（】・6x40cm）w九1Chwasequ班brated with50mMphosphatebuffer（pH6．8）comtalninglOmMMgC12．

ThegelchfOmatOgraPhlCelutionpattem（fractionrange： ．

indicatestheelutionpatternsofBlueDextran（voidvolume，m．W．2xlO6），VitaminB12 （m．w．り50），Zn2十andCo2＋血ichwere那edaざCdibrat血5加dardg．For仏emjddle graph，the proteinconcerLtrationwasdeteminedat280nmandalsobythemethodof Ij）Wry；the concentrations of Cu，Co and Cd for each fraction were determined

by atomic absorptlOn・Thelower graph showsthe elution pattem ofZn，Mn and Fe

－8－

TableI．

HeaYyMetalContentsoftheLeavesofVariousPlantSCollectedfromThreeSites3）

Metalcontemt hg／gdけW¢iか）

Site Sp¢Cies Mn Zn Co Ni Cd Fc C11

Ginzandaira■ C毎J朗〃e〝fJ e．∫e〝甘ね

e．椚0〃gOJね〃

ヱc〟岬f血血m

」．γ0わ∫Ce那e

60302856羽

3

0 0 0 00＋′n

5 2 5 7 8

3 3 3

8．0

＜0．5

＜0．5

＜0．5

＜0．5

7 5 0

2 5 つ】 7 0

1 0 0 0 2

7

つ】 つ‘つ】 qノ 4

0 1 つ】 （U O

つ】 ∠U ▲4 1 7

7 0 0

0 つ】・J 5 0

8 0 ＜U O つん

4 つ】 4 9 4

′n▼ 轟′－（′ O l

Mikouchi■ C丸山蜘即血

e．ぶe〝βね

e．mo〃gO肋8

1 7 つJ

′b▼ つ‘▲J

9 7 3

0 0 0

3 2 〈ノー

「－ 1 ・・－

／b 7

ノ人U 7 「‘ 7 0 0

穴Ⅳj

つ】 つJ つJ

Nikko… Cゐ〃rわ加e〝fざ

e．Je〝甘ね

e．椚P〃gP肋〃

ヱcI上申抽出椚

A．γ0わざぐe〃ゴビ

0 ∠U O つ一5 舶4476

（U O O O O

qノ 00 4 7 （1．〇

l つJ 2 1

0 7 7 5 4 くJ一7】 （∠ ′h▼ rJ

1 0 5 0 4

つ】 つ】 2 つJ （ノー

（U 7 0 0 0

1 7 つ▲ 5 3

1 1 1 つ▲

0 4 5

′0 2．．1 4 5 ′」U O O 1 0

／D 2 つ一〇 3

5 つん （∠ l つん

19

＜0．5

＜0．5

＜0．5

＜0．5

CゐαJ朗〃e〝由

せ、∫l、〝甘几

e．mo〃gOJ如

上蝕画加肋 A．タ0加∫Ce〃∫e

C7eJゐ′Wムαrゐ加e〝由Sieb．eJZucc．

e〟gmJJer招ねmunb． e〟erC〟J椚0〃g（フJfcαFisch．Ⅴ打．卯∫∫eJg耶ね

Rehd．eJW鮎． タ0如〃〟mC〃甲山血血椚Sieb．eJZucc．

Arわ′血mγ0わざCeJIJemdst．

■ Ashiodistrict，TochigiPrefecture． ＊■ Nikkobotanicalgarden，Tochi由Prefecture

－9－

Z01ト岳OS凸く

10 20 30 40 50

0 10 20 30 40 50

FRACT10NNUMBER

Fig．1，Elution pattem ofthe buffer extIaCt frompePperbushleaves on a Bio・GelP4 COlumn（1・6x40cm）equnibratedwith50mMphosphatebuffer（pH6．8）containing lOmM MgC12・HighproteinconcentrationsmeasuredbyLowry’smethod（middle， fraction No．30－5q）are duetothe reactlOnbetweenFolin’sreagent andthelow molecularpigments．BlueDextranyitaminB12，SOlutionabsorption．Metals，atOmic absorption．Solidline，280nm；Brokenline，650nm，

－10一

extractedfrompepperbushleavesonthecolumn・Fig・1indicatesthatpossiblemetalsbound tohighrr101ecules（moleculeswhichhavemolecularweightmorethan．4000areelutedat YOidvolume）includeCuandZn，thoughrnostofthemetalsweredetectedattheelution volumeoflowmolecularweightspecies．AsforCo，CdandMJl，theelutionvolumeofthe metdswasalmostthesameasthoseofCopandZnions，SuggeStingthatCo，CdandMnh pepperbushleavesarepresentaslowmolecularcompoundsorions・

Furtherpuri爪cationofcobaltLCOntainingcompound（S）wasperformedbyionexchange chromatographyonaDEAEandaCMce11ulosecolumnbutthepresenceofasinglecobalt SpeCieshasyet to be demonstrated・Fromtheelutionpattemsofgelfiltrationandion exchange chromatography，CObaltin pepperbushleaves appears tobe present as alow molecularweightcompound（s），POSSiblyacobalt・pigmentcomplex，and／orascdbaltions・ Furtherstudiesarerequiredtoidentifythechemicalformsofcobaltinpepperbushleaves・

ヱAα押er伽Je加／蝕わ叩α′血ノ血加ed伽∽♪申Per如力⊥e〃？e∫

AsshowninFig．1，partOrthecopperinpepperbushleavesisboundtohighmolecular Weightcompounds．Thepurification ofthecopperproteinwas，therefore，perfomedand SOmePrOpertiesoftheisolatedcopperproteinaredescribed・

Plan（material．LeavesofpepperbushwerecollectedinAngust，1977，atMikouchiinthe AshlOdistrict，Tochi由Prefecture，WherelargeamOuntSOftheleavesarereadilyavailable7）． Theleaveswerewashedwithdeionizedwaterandstoredat・20Cunt山required，

hJr押ation．Unlessstatedotherwiseallstepswereperfomedirlthecold， Stepl・＆tTqCtion・FfOZenleavesofpepperbudl（200g）weeCutintosmallpiecesand groundinamechanicalmortarfor30minwith50gofpolyvlnylpyrrolidone（insoluble）， 200gofsandand700mlof25mMTri・HClbuffer（pH8．0）containinglmMdithiothreitol． The homogenate wascentrifuged forlOrnin at3000xgand the sedimentwas hlrther groundfor30minwith700miofthesamebuffer．AftefCentrifugationofthehomogenate， thesupelnatantSOlutionwascombinedwiththatobtainedbythenrstcentrlfugation．Four－ teenadditionalbatches oftheleaveswere similarly treatedandthesupernatantsolutions werepooled（151iters）．

Step2．Ammonium su埴tef相C（ioTZation．The supernatant solutionwasbroughtto30％ Saturation withsolid ammonlum Sulfate．After standlng forlhr，the preclpitate was removedbycentrifugationfor15minatlO，000xg二Thesupematantsolutionwasbrought to90％saturationby山rtheraddition ofsolid ammonium sulfate andle丘standingover－ night・Theprecipitatewascollectedbycentrifugationfor15minat10，000xg，SuSpendedin anddialyzedagainst25mMTris－HClbuffer，pH8．0．Thedialyzatewasclarifiedbycentrifu－ gation and brought againto90％saturationwithammonium sulfate．Theprecipitatewas COllectedbycentrifugatlOn，dissoIvedinanddialyzedagalnSt25mMTris・HClbuffer，PH8．0・ Step3．St甲WtSeDEAE・CeL）uLose chTOma（OgnPhy．The dialyzedsolution（370mi）was appliedtoacolumn（2・6x35cm）ofDEAE－Ce11ulose（WhatmanDE－23），previouslyequili・ bratedwith25mMTris・HClbuffer，pH8，0．Thecolumnwaswashedwlththesamebufferto 由VeaneluateabsorbancevalueoflessthanO．1absorbanceunitsat280nm・Subsequently， 也ecd11mnWaSelⅥtedwi也400mlorユ5mMTIis－HClb甘汀由，pt18．0，COntalnin各0．4M NaCl・The protein fractions whose Cu concentratlOnSWeregreaterthanO・lJ］g／mlwere pooledanddialyzedagalnSt25mMTris・HClbuffer，pH8・0・ 岳tep4．Lineargndien［DEAE－CeLLulosech′OmatOgTaPhy（Fig．2）．Thedialyzedsolution （180ml）waschargedtoacolumn（1．6x30cm）ofDEAE・Ce11ulose（WhatmanDE・32）， PreVlOuSly equilibratedwith25mM Tris・HClbuffer，PH8．0・Elution oftheproteinwas

－11－

Cu， 刃g／mt

Fig・2・ChromatogIaphyonDEAE－Cellulose（DE－32）．Fordetai1s．seetext．

，aeat28 ▲‾・ ，COpper（atomic abs。，， c。nCent，a．i

Fig・3・SecondChromatographyonSephadex G・75・Fordetails，SeeteXt． 0－0，a♭50rb且mCeat280几m； △－△ ，absorbancea【600nm； ・L－一一，COpper（atomicabsorption）．

－12－

accomplishedwithalineargradientpreparedbymixingl00miof25mMTris－HClbuffer bH8・0）contalningO・04MNaClwiththesamevolumeof25mMTris・HClbuffbr（pH8．0） eontainingO・4MNaCl・AsshowninFig．2，thecopperprotehwaselutedataconcentration

Ofabout O・2MNaCl・The fractionsindicatedbythehorizontal1ine（CuconcentratlOnS greaterthanO．］pg／ml）werepooledanddialyzedagainstdlSti11edwater．

Step5・GetPtntiononSephodexG－75（Fig．3）．Thedlalyzedsolutionwaslyophilized andtheresultantyellow－gree血血powderwasdissoIvedinlOmlof25mMTris－HClbuffer， pH8・0・Thesolutionwasappliedtoacolumn（2．6x65cm）ofSephadexG・75，previously equilibratedwiththesamebuffer・Duringelutionwiththebuffer，afhlntbluebandwas Observedunderanorange・COlouredbandandthisbluebandcorrespondedtothepeakofthe COPperprOtein・Inthisp－OCedurethecopperprOteinwasnotcompletelyseparatedfromthe Orange・COloured pigment・ThefractionscontainingmorethanO．1rFLg／mlofcopperwere， therefore，againappliedtothecolumnofSephadexG－75andelutedwiththesamebu胎一．

AsshowninFig・3，theabsorptionpeaks，A280andA600，andthecopperpeakwerecoinci－ dent・The fractionsindicated by the horizontal1inewerepooledandusedforanalyses describedbelow，

1bmogeneiO，・Polyacrylamidediscgelelectrophoresisgaveasingleproteinband（Fig．4）．

舶油肌血Ⅷ頑姉川血虻血抑姉ノ．

1・Themin血ummolecularweightwascalculatedfrom山ecoppercontent．Theprotein COntainedO・75％copper，WhichgaveaminlmummOlecAnrweightof8500．

2・Themolecular weight wasestimatedbygelnltratlOnOnaSephadex G・75column （1・6x95cm）equilibratedwith50mMTris－HClbuffeI，pH8．0．Thestandardsusedfor Calibration were cytochromec，ribonulease，myOglobin，Chymotrypsinogen and oval－ bumin・Themolecularweightofthecopperproteinwasestlmatedtobeapproximately 17，000（Fig．5）．

3・ThemolecularweightofthesubunitsoftheproteinwasestimatedbyacTylamidegel electrophoresisinthepresenceofsodiumdodecylsulfate，withandwithout2－merCaptO・ edlanOl（1％wv－1）．AsshowninFig．6，theproteinwasnotdissociatedbysodium dodecylsulfateunlessmercaptoethanoIwaspresent．Theprotein，incontrast，WaSdissoci－ atedintotwosubunitsofequalsizeinthepresenceofmercaptoethaTLOl・Usingcyto－ Chromec，myOglobin，ChymOtryPSinogen andovalbuminasthe molecular weight Standards，the molecularweightsofthe proteinandthesubunlt WereeStmlatedtobe about20，000andlO，000，reSpeCtively．

Absorption甲eCtrum・TheproteinisolatedbytheproceduredescribedaboYeWaSfhintly blueincolor・Theproteinturnedmoreintensebluebytheadditionofafewcrystalsof fe’rrlCyanide，Thebluecolorwasbleachedcompletelybyfurtheraddltionofsodiumascor・

bateandawater．clearsolutlOnWaSObtalned・AsshowninFig・7，inthevisiblerange，the PrOteinintheoxldlZedformhadabroadpeakmaximumat600nm・］ntheultravioletrange itshowed，besldesthemainabsorptionpeakat278nm，thecharacteristicpeaksat259，265 and269rLmCOrreSPOndingtothe血estruCturebandsofphenylalanine，andashoulderat 284rLmCOrreSpOndingtothatoftyrosine；thespectrumWaSVerySimnartothoseforspinach plastocyanin8）andtobaccoplastocyaJlin9）．TheA600／A278ratioofthiscopperPrOteinwas O・25andismuchlowerdlanthoseforspinachandtobaccoplastocyanln（A597／A278ratio Ofthepurinedplastocyaninareaboutl．0）．

Me（alcontents．Thecoppercontent ofthe purinedproteinwasdeterminedby non・

flameatomicabsorptionspectrophotometrywithaPerkln－Elmer503speCtrOmeterntted WithaHGA2000atomizer・Sin1ilarlyzinc，manganeSeandironweredeterminedbyflame

－13－

一 － ■、－

ご こ；去ぶ、

－

一顧

● ＋

●こ一

Fig．4

Fig・4，Polyacrylamldediscgelelectrophoresisofthepurinedcopperprotein・Theprotein ・（50pg）wassubjectedtogelelectrophoresis（ge17・5％，PH9．5）for40minat4mA pertube．Thegelwasstainedwith1％amidoblacklOBin7％aceticacidand dest血edin7％aceticacid．

Fig・6．Sodlum dodecylsulfatepolyacrylamidegelelectrophoresisofthepurinedcopper protein．Theprotein（1qLLgeaCh）wassubjectedtoelectrophoresisonpolyacrylamide gel（1昭）inthepresenceofsodlumdodecylsulfate，with（right）andwithout（1eft） 1％2－merCaPtOethanol，aSdescrlbedbyWeberandOsbomll）．Thegelswerestained with0．1％CoomassiebrilliaJltblue（aeeticacid，1：methanol，5：distnledwater，4）foI 60minandexcessstainwasremovedusing7％aceticacid．Itisapparentthatdissoci・ ationintosubunitswasachievedonlyinthepresenceof2－merCaptOethanol，

－14－

l．2 4．1 4．1 l．8 5．1

LOG MOL．WT．

4．l

Fig．5．Estimationofthemolecularweightofthecopperprotein丘ompepperbushleavesby gel瓜1trationonaSephadexG－75column（l．6Ⅹ9．5cm）．Themolecularweight Standardsusedwere：1．cytochromec（12，400），2．rlbonuclease（13，700），3．myo・ globin（17，000），4．chymotrlpSinogen（25，800），5．ovalbumin（43，000）．Theclosed Symbol（theelutionvolumeofthecopperprotein）correspondstoamolecularweight Of17，000．

一

l

● l

OUu■モ○仇一く

脚 脚 Ⅵbv0longth 210 仰 ：は0 110510 551 （nm）

Fig・7・UltravlOlet and Yisible absorption spectrum oftheisolated and purined copper proteh・The protein concentration was O．69mg／mlin25mM Tris－HClbuffer， pH8・OLTheabsorptionspectrumwasrecordedonaHitachi323spectrophotometer・

ー15一

atomic absorption spectrophotometry withaHitach1508Aspectrometer・TheresultsiTI TableIIindlCatedthattheproteincontairLedtwoatomsofcopperPermOleculesuggesting thatoneatomofcopperwaspresentperOneSubunit・NosignificantamountofzirLCCOuld bedetectedintheprotein・Thecontentsofironandmanganesewerebelowthedetection 近mit50ト血ere5匹C加eatomicabsorpt血tec血jques・

EnzymLcactivio，．SupefOXidedismutase（ECl，15．1，1）activitywasassayedaccordingto themethodofMcCordandFridovich10）buttheproteindidnotexhibitenzymicactivity．

Amlnoacidan（砂Sis．ThecopperproteinwasdialyzedagairLStdlStilledwateTforaweek．

TriplicatesamplesofO・7mgoftheproteinwerelyophilizedinaeid・WaShedtubes，dissoIved inlmlofconstant．bo揖ngtlClandsealedundervacuum．Hydrolysiswasaccomplishedat llO℃for24，48and72hr・AminoacidanalysiswasperformedwithaHitachi835amino acidanalyzer・Therecoveriesofserineandthreoninewereextrapolatedtozerotime．Ⅴaline andisoleucineweredeterminedfromtheYaluesat72hrofhydrolysis・TablellIpresentsthe resulton amiJlOaCidanalysisofthecopperprotein丘ompepperbush，andcornparesthe resultwiththoseofspinachplastocyaninbyKatoheta］8），Theaminoacidcomposition Showed considerable similarity to that ofspinach plastocyanln，thoughthecontentsof asparticacid，glutamicacid，glycine，medlionineandlysinewerelower．

乃bleII．

MetalContentoftheCopperProteinIsolatedfromPepperbushLeaves．

AftertheproteinwasdialyzedagalnStdlSti11edwaterfor2days，themetalcontentswere determinedbyatomicabsorptlOnSPeCtrOmetryandtheproteinconcentrationwasdeter－ minedbythemethodofLowryetaL12）usingbo血eserum山bumin且SthesfandBId．The mole／moleratiowasobtainedonthebasisofamolecularweightof17，000．

Metal content

Metal Flg／gprotein g・atOmSmetd／moleprotein

CuZnFeMn

2．0

0．06

below O．01

below O．01

ー16一－・

TableIll．

Amino Acid Composition

TheamlnOaCidcompositiorlbfthecopperproteinandcomparisontothedataforspinach Plastocyanln・Al1calculationswerebasedonamoleculaTWeightof17，000forthecopper prote血．

Pepperbushcopperprotein Spinachplastocyanin8） AJnil】O aCid

Residuespermoleculeofprotein

Aspartic acid

nreonin（〉

Se血e

Glutamic acid

P101ine

Glycine

Alawe

V姐ine

Methionin（∋

Isoleucin（，

Leucine

Tyro血e

Ph¢nylalanhe

lザSine

王事stidine

A一群Ihe

HalfヾyStme

Tryptophan

2qノ0 ′0／b 凸ノ3 ′04 0ノ′0∩フ5 2201日l

′○ ／b▼ てJ つ】 0ノ 5 0ノ 5 2 3 0ノ つJ q／ 7 3 つ】

76842702

．

l

l▲

つ人－1 11 つ】 つん 1⊥ l

a）notdetem血ed．

〃．用γJわわg加J尺oJeo／CbわαJrわ′タ甲pe′毎ざ力月如上・

An0therinterestingphysiolo由CalaspectrevealediJITablelisthepossiblenutritional sigmincanceofthehighcobaltcontentinpePperbu血plant．Ageneralrequirementforcobalt byhigherplantshasnotbeenestablished，eXCeptわrnitrogen爪xationbythesymbiotic root・nOdulebacteria13，14）．ItisofinteIeSt，therefore，tOStudywhethercobaltisessential 払rpepPerbu血plantornot．Wetestedfortheessentiality，tOXICityanduptakeofcobaltby varyingthe amounts ofcobalt added（none added to10ppm）toculture solutions・h addltion，CObaltwasaddedaseithercobaltsulfateorvitamineB12tOeXarrunetheefftctof chemlCalfomsofcobalt．PepperbushplantsgrownfrolnCuttingswerecultivatedinsolution inatemperature＜OntrOlledgreenl10uSe・Fig・8showsthephotoofpepperbu血plantsafter about6weeks，cultivation．ItwasfoundthatdleCObaltuptakebytheplantremarkably dependedonthechemicalfomofcobaltadded，andthatthecontentsofironandmanga－

ー17－

nese¢裏11bitedamarkeddependenceonthecobaltconcentTations肌d仙echemicalfomsof CObaltintheculturesolutions．Thisworkwillberepoftedindetailelsewhere，

肇行李賛

‘ ． こ・ 1こニー 丁 ′－

寮狩

None Added－

0 ．ニ‥．、

Fig．8′Pepperbushplantsinsolutioncultureafterabout6weeks’cultivatlOn・Pepperbush plantSgTOWnfromcuttingswer¢Cultivatedintheculturesolutions15）towhich co♭altw且Sadded且SeidleTCObaZtsu）Eate（upperserjes）orvltaminB12（Jowerseries） withCoconcentlatiollSran痩ngfromヱerOtOlOppm・

－18‾

Referemces

l・Yamagata，N，＆MuTakami，Y．（1958）：Acobalt－aCCumulatorplant，C7ethTabarbherv由 Sieb．eJZucc．抽出re181：1808・1809．

2・Yamagata，N．＆Murata，S．（け糾）二Accu汀l血ti甜Ofcobal暮卸dtllepre5enCeDrCOb山・ porphyrlnCOmplexinC7ethTa，ahigherplant・ButLhst・PubLHealth・13：170－175・

3．Okamoto，K．，Yamamoto，Y．＆Fuwa，K．（1978）：Accumulationofmanganese，Zinc， cobalt，nickelandcadmiumbyC7ethnbaTbinen，is，Agric・BioL・C7zem・42＝663・664・

4、Yamasata，N．（1978）：Bioaccumulationofheavymetals・KugzzktLtOKogyo31：957・ 960（mJapaTleSe）．

5．Kondo，N．＆Sugawara，K（1978）＝Studiesonresistanceofplantstosulfbrdioxide（4）； su］LiteoxidatlOLlfeaCtionsinhigherplants．1n”Studiesonevaluationandameliolation ofalrPO11utionbyplants，，・Progressreportin1976－1977・Reportofspecialresearch prq】eCt，NIESR－2．

6．Cannon，H．L．（1960）：Botanicalprospectlngfbroredeposits・Stienee732：59l・598・ 7、Okamoto，K．，YamamotoY．＆Fuwa，K．（1978）：Pepperbushpowder，aneWStandard

相良代nCe汀1aterld．A鵜止C翫Ⅶ．50ニ1950－1951．

8．Katoh，S．，ShiIatOri，I．＆TakamiyaA．（1962）：Purin印tionandsomepropertiesof SPinachplastocyanin．］．Biochem．（Tokyo）51：32－40．

9．Schnlld，G，H．，Radunz，A．＆Menke，W．（1975）：TheeffectofanantiserumtOpla＄tO・ Cyaminonvariouschloroplastpreparations，ZNatuゆrgch．30C：201－212．

10，McCord，］胤＆Fridovich，1．（1969）：SuperoxidedismtltaSe，anenZymicfunctionfoI ery血TOCupreh仇emocuplei叶エβわJ．α1em．244＝6049・6055・

11．Weber，K．＆Osbom，M．（1969）：Thereliabilityofmolecularweightdeterminationsby dodecylsulfate－POlyacrylamidegelelectrophoresisJBioL．C77em．244：4406－4412・

12．Lowry，0．H．，Rosebrough，N．］．，Farr，A．L＆Randal1，R．）．（1951）＝ Proteinrneasure－ mentwiththeFolinphenolreagent．］．BわLC7zem．193＝265－275・

13．Ahmed，S．＆Evans，HJ．（1959）：Effectofcobaltonthegrowthofsoybeansinthe absenceof5uppliednit∫Dg印，βわ仁方e仇βわpJぴ∫・尺e鼠Cb肌用肌1：27ト275・

14．Reisenauer，H．M．（1960）：Cobaltinnitrogennxationbyalegume・NohJre186：375－ 37（i．

15．Epstein，E．（1972）：Themediaofplantnutrltion．h‘‘MineralnutritiorLOfphnts： Principlesandperspectives”．（JohnWileyandSons，Inc・）p．39・

－19－

CHAFrERI‡1

PREPARATIONOFPEPPERBUSH

K．Okamoto

ThepreparationofPepperbushconsistedofthefollowingsteps．

助叩血勘抑㍑吋極

Theleavesofpepperbu血wereco11ectedinSeptembeT，1975，atMiko11ChiintheAshio distrlCtWherelargeamountsoftheplantarereadilyavai1able，Theleaves，freefromstems， WereWaShedwithdeionizedwaterarlddrledinanairovenat80℃ovemight．About30kg OfthedryleavesweretlSedinthiswork．

Grl円d如g¢円dぶfe血g

med∫yleaves（about700g）weregroundforaboutonehourinabal1－mnl（95％A12O3， 71iters）whichhadbeenpleviousbgTO11ndwellwiththeleavestominimizecontamination bymetal・Thepulverizedsampleswereplacedonasetofsieves－a50－meSh（297LLm）nylon SieYe（top），a80・meSh（177JLm）nylonsieveOT）iddle）・BndaTeSerVOjTmadeofvinYIchloride （bottom），andthenvibratedmechanical1yfor15min．

Jい・r・ト・ごご叫・ご，～fl、・J′∫・バい一′ト′バ．ト・仙川J・い′トJい・いり・、いじ、

Todeterminethedistributionandvariationofmetalcontentforthesamplesdiv］ded加O the叫eepa－tS（above50－rne血，fTOm50to80－me血，below8D－meSh），nVelotsoftheleaves （about700geach）wereindependentlygroundandpassedthroughasetofthesieves．Three portions（lgeach）were taken fromeachofthethreepartsinfiYelots，dry・aShedand analyzed・Zn＞FeandMnweredetemlinedbyatomicabsorptionspectrometryusinganair－ acefy】el】8鮎me．

Tat）1eI．

DistributionandValiationofZn，FeandMnわrVariousMeshSizes（LotNo．3）

Contemtd，〃g／gdけWt．， Rel．std．dev．，％

Zn Fe Mn Zn Fe Mn

2．6 3．2 1．2

1．5 2．3 0．3

0．5 0．5 0．5

Above50・me血 335 87 1950

50to80－meSh 331 97 2030

Below80－meSh 301 152 1950

αaVerageV

－20－

TableIshows，forexample，theandyticaldataonLotNo．3（theanalyticaldataonthe otherfourlotswerealmostthesame）．The爪mestpart（below80－meSh）containedlessZn andmoreFethantheothertwoparts．Mnlevelswerealmostthesameforthethreeparts． Thesedifferencesinthemetalcontentsofthevariousfractionsmaybeeonnectedwiththe biochemicalrolesofthemetals．

Therelative standarddeviations forthemetaldeterminationsarealsoglVeninTableI・ Thefinestpartshowedthelowestvalues，indicatingthebesthomogeneity，thoughthevalue ofMnisalittlehlgherthanthatforthe50to 80・meShThesamplespassedthrougha 80・meShsievewere，therefore，Selectedforfurtherprocesslng，

．け血†lg

Thepowderhavingpassedthroughan80－meShsievewasdividedintotwopチrtSWitha rifflesampler（JISNo，2）madeofvinylchloride．ThepowdeTWaSPiledupintwolayersand againdividedbypassingthrbughtherifnesampler．Pepperbushsampleswerehomogenized byrepeatingthisproceduretentimes．

現厨川／〟加ゎgw油α尺脚e5b椚〆er

Thehomogeneity ofthe samplesobtainedbymixlngwitharifnesamplerwastested・ Beforemixing，threeportions（1geach）weretakenfromeachofthe丘velots（320，210，420， 320and320由，dry－aShedandanalyzed．＾fterthenvelotsweremixedbypassingthrough therifnesamplertentimes，nineportions（1geach）weretakenfromthemixedpowder，dry． ashedandanalyzed．

Tablellshowsthe analytlCaldataonthepepperbushsamplesbeforeandaftermixlng・ Beforemixing，therelativestandarddeviationsofZn，FeandMndeterminationsinthenve lotswere6．2，12and13％，reSpeCtlVely，After mixing，those ofZn，Fe andMngreatly decrea，Sedtol．0，3．1andO．7％，reSpeCtively．Goodhomogeneitywas，therefore，aChleVedby mixingwiththerlmeSampler．

TableII．

EffectofMixingwithaRlffleSampler

Before miving After mixing

Zm Fe Mn Zn Fe Mn

FJg／gdrywt． FLg／gdrywt．

MaxlmumValue 340

Minimumvalue 290

Average 309

Rel．std．dev．，％ 6．2

0 0 0 3

′人じ っJ n7 1 4 7 9

2 1 1

l つん ／」U O 302929L

ヽJ 1 7 1

1312123．

70405〇

．7 0 0 0 0

つ▲一ソ】 （∠

つん 0 ハB つん

5 1 つーl

〟0椚0邸〝e砂qげ申pe′ム〟∫わ助椚〆eJO〝α上α堵e∫c〃Je

Sevenlots（total14，4kg）ofthepeppeTbushsamples（below80－meSh）weremixedinthe rime sampler（ten times）andthepowderwasrandomlypackagedinl150glassbottles （about14geach）．Tochecktheho血ogeneityofthepepperbushsamples，38sampleswere taken fromeachof30bottles，dry－aShedandanalyzed・Homogeneitywasestimatedfrom

－21－

therelativestandarddeviationsofZn，FeandMndeterminationsofthepeppeTbushsamples・ TableITIshowsthattherelativestandarddeviationsforZn，FearldMninthe38samples

Werel．5，2．2andl．0％，reSpeCtlYely．WhenstandardsolutionsofZn．FeandMnatthesame COnCentratioIISaSthesamplesweremeasuredtentimesundersimilarconditions，therelatlVe Standarddeviationswerel．4，2．7andl．0％，reSpeCtively．mevariationofthemetalcontents of the pepperbush samples were almost the same as the corresponding results for the Standardsollltions，indicatingthatthepreparedpepperbushsamplesarehomogeneousand pTaCtlC劇yusef山asastandardreferencematedd．

TableIII．

Homogeneity of Pepperbush Samples

W

eⅣ F d

g ．即 〃

10209〇．〇

1 0 （U 1 2 つん つん

2 1 ′b つん

716162．

1

Maximumv山ue MinimllmV山ue

Average

Rel．std．d8V．，％

（38smples）

5 5 7 5

343233L

R£1．std．dev．，％

（5td．5dutjoJ110time5）

Reねremces

Okamoto，KリYamamoto，Y．＆Fuwa，K・（1978）：Pepperbushpowder，aneWStandard referencematerial．d〃〃J．αe椚．50：1950・1951．

Okamoto，K．＆Fuwa，K．（1979）：PreparationofenviroIlmentalstandardreferencematerlals． ∬〟〃砂∂5b血血iG陶助6（4）：1・6（inJapanese）．

ー22－

Aluminaba山一m山

Sieving apparatus

－23－

鮎mesmpleT

凝議燦綬鷲オ

紛漆涙

：、 ・ ＼（！．1

i－Ⅰ汗IJiこtミIit：Sl王 l．1ヽLち

ヽ＼加ヽtlらヽ、lけ 、 r l■、、

√ ■ 、 ■ jliンl｝

PepperbushStandardReferenceMateIial

－24－

CHAPTERIV

ANALYSISOFPEPPERBUSHBYATOMICABSORPTIONAND

FLAMEEMISSIONSPECTROMETRY

K．Okamoto

加古タロducJわ〃

Atomic absorpdon spectrometry（ÅAS）is a uniqeanalyticaltechniqueforthe determinatlOnOfmetal1icelementsandisusedinmanysclentific鮎1ds・Theacceptanceof AASisattributedmainlytothefo1lowingfactors＝1）highsensitivityforalargenumberof elements，2）abilitytocontrolandovefCOmematrixinterference，ifpresent，3）speedof analyses，raPidimprovementininstrumentatiorl，andsofor止L

TheatomicabsorptlOnteChniquecanbegroupedintocategories＝（a）爪ameAAS，（b）non・ flameAAS，Withrespecttothemodeofproducingtheatomicvapor．rllStOrica11y，aname hasbeenthemeansofatomizatlOnforAASarldissti11thebasicsourceforthevastma」0rity OfAASmeasurements，becauseoftheadvantagesindicatedabove．However，disadvarltageS arethat arelativelylargequantityofsaJnPleisrequiredandsensitivitylSlimitedformany elements．

A relatively new techique for producingtheatomicvaporisthenon・鮎medevices・ Thesedevicescanbegroupedintocategoriessuchas（a）thecathodicsputteringtechnique （b）8raphlte fuTnaCe Or CaTbon rod devICeS（e）tantalum rlbbonand（d）thecdldvapor method，Thesemethodsdifferfromflamesinthattheatomisationsourceismoreconducive to the productlOn Ofground state atomsin contrast tothe chemical1y reactlVe name environment・Also，forthegraphitefumaceorcarbonrodonlyarelatlVelysmal1volumeof thesample（1to50FLl）isnecessaryforanalysiswhich辞VeSthesemethodsimprovedsensi－ tivityoYernamemethods・However，thenon．鮎memethodshavetheinl1erentdlSadvantage inthathigl1PreCisionismoredimculttoattain．

ThesamplessuitableforAASanalysiscoverawiderangeofmaterialsandconcentrations． The procedureforsamplepreparationdependsonthetypeofsamples，theelementstobe measuredandtheprecisiorlandaccuracyrequired，therefore，thereisnogeneralmethodfor thepreparationofal1samples・Solutionsamplesarenebulizeddirectlytothenamebutfor SOlidsamplesadissolutioninhydrochloric，nitricorpercllloricacidsisfirstrequired・Proper pretreatmentofsamplesismost血portanttoperforlTLAAanalysis．Thesamplepreparation forPepperbushis，therefore，descdbedhereandalsotheanalyticalvaluesforpepperbushare presented．

J・助叩血伽御閥触りれ伽血血加ゎ（オ∬，仇， 〟g，〃〃，ZJ†，魚，〟α，月∂，（b，仇，朗α〃d（U．

Thefo1lowlngprOCedure＊hasbeenappliedfordissolutionofPepperbushpowder．Aone

暮 AdaptedfromT．Rainsl）．

－25‾

OltWO gram Sample，pIevioudy drled at85 ℃for4hrsinanoven，WaStIanSferredtoa tenonbeaker・Next，HNO3（20ml，l：1）wasaddedandthesystemwaslenstandingover・ night．Thebe独erwasheatedonahotplate（200 ℃）foraboutanhour，COOledandthe SidesofthebeakerweTerinsedwithdistil1edwateI．AfterhlrtheradditlOnOfHNO3（10rnl） andHF（5ml），仏esolutionwasevaporatedtoapprox皿ate】ylOmlby九ea血g．Wheれblack Particlesofunreactedcarbonsti11remained，theaddjtionofHNO3（10ml）andheatingwere repeated・AftertheadditionofHClO4（10ml），thesolutionwasheatedtostrongfumesof peTCh］oJjcacjd，COOledandthesidesofthebeakerwerewa血edwithdistilledwater．Heating WaSContlnueduntilthesamplevolumewasreducedtoapproximately2mi．Thebeakerwas leftstandingforaboutonehouraftertheadditionofapproximatelylOmlofdlSti11edwater・ Thedigestwastransferredtoal00mlvolumetricflaskanddilutedtovolumewithdist山ed Water．ThesamplesolutionwasstoredinalOOnlpolyethylenebottle．

2∴弘椚pお丹印α和め〃舟r

Jゐeβeナビ〝〝およわ〝β′乃．

Acloseddigestionsystemi11ustratedinFig．lwasadoptedforthedetenTunationofPb． This systemis preferred when dealingwith volat址e elemeTltS．OnegramofPepperbush powder（れOt driedin an oven）wasplacedina鮎sk（A）contai血g5mlofconcentrated HNO3andleftoYemight，Thenaskwasnttedwithacondenser（B）andanacid－trap（C）to Which5mloflMfTNO3WaSadded．Onemlof30％H202WaSaddedtotheflask（A）each dayfor4consecutivedaysandgentleheatingonagasburnerwascontlnueduntnthedlgeSt becameclear．ThedigestandtheHNO3inthetrapwerequantitatlVelytransferTedtoa100 mlvolumetric tlaskand the solutionwasmadeupto dlemarkwithdlSti11edwater．The Samplesolutionwassubsequently丘1teredthroughaMi111pOrenlter（25mm，0．45FL）which 血adbeenple扇ouslycleanedinO．1MHNO3．

j．伽re〝乃加〃rわ〃（げ〟，GJ，埴，肋，ZJl，爪，，肋，尺占，Cb，

Cわ，」Ⅵβ〃d（ガわ′dJo朋ねd如p甲fわ〝勘）eC加∽efワ

乙ねわgAfrrAce伊Je〝e月αme．

PreparationofStandardStockSolutions

Standardstocksolutions（1000ppmofeachelement）werepreparedeitherbydlSSOIving highpuritymetalpowdersin appropriateacidsorbydissoIvlnghighpmitymetal－Saltsh distilledwater，aSShowninTableI．

Standard Working Solutions

AseriesofindividualstandardworkingsolutionsforMn，Zn，Fe，Co，Cu，NiandCdwere preparedbyapproprlatedilutioTISOfthe1000ppmstocksolutjonswithO－1Nf†C］04． rangesofthestandardworkingsolutionwerechosentoencompasstheconcentrationsinthe Pepperbushsolution．ForNaandRbdetemlinations，Kwasadded（nnalconcentrationofK was150ppmwhichcorrespondedtotheKleYelinthesamplesolutions）toeachofstandard workingsolutionstocompensateforionizationinterferenceeffects・Forthedeterminations ofK，CaandMg，thedigestswerefurtherdilutedfiftyt血eswith0・1MHClO4・Aseriesof mixedK，CaandMgsolutionswereusedforstandardlSation・

－26－

TableI．

StandardStockSolutionsforAtomicAbsorptionSpectrometry■ 1000FLg／mi（ppm）astheelementina丘nalvolumeof500ml．

Ⅲement Procedure

DissoIveO．9534gKCl（99．999％）indlStnledwateranddilutetovolume， Transferl・2487g CaCO3（99．999％）in500mlvolumetric・naSkwith 100爪Iwater，add5mlHCl，afterCO2isreleased，dilutetovolume， DissoIve500mgMg（99．9％）inHClanddilutetovolume． DissoIve500mgMn（99．8％）irLHNO3＋HClandd山tetovolume． DissoIve500mgZn（99．99％）inHNO3anddilutetovolume． DissoIve500mgFe（99．98％）inHNO3十HClanddilutetovolume． DissoIvel・271gNaCl（99，999％）indistilledwateranddilutetovolume． DissoIveO・7074gRbCl（99．99％）indistilledwateranddilutetovolume． DissoIve500mgCo（99．99％）inHNO3＋HClanddilutetovolume． DissoIve500mgCu（99．99％）inHNO3anddilutetovolume． DissoIve500mgNi（99．99％）inHNO3anddilutetovolume． DissoIve500mgCd（99．99％）inHNO3anddilutetovolume．

MgM。Z。FeNa馳C。CuNiCd

ExperimentalConditions

TablelI．

ExperimentalConditionsforDeterminationsofK，Ca，Mg，Mn，Zn，Fe，Rb，Co，Cu，Ni andCdinPepperbushSRMbyAtomicAbsorptionSpectrometry

UsingAir－AcetyleneFlame

Element Analytical hmp FlowRate Bumer Background llne Current Slit Width Air－Acetylene Height Correttion

（m） （mA） （Å） （1／min） （mm）

KCaMgMnZnF。Na馳C。C。NiCd

769．9

422．7

285．2

279，5

213．8

238．3

589．6

780．0

240．7

324．8

232．0

228．8

8 （古 （古 qノ 8 qノ 8 8 qノ 8 qノ 00

3 3 3 1 3 1 3 3 1 3 ▲1 3

10－2．5

10・2．5

10－2．4

102・6

10－2，4

10・2．5

10－2．4

10・2．4

10・2．5

10－2．3

10・2．3

10－2．3

4 （U 5 4 4 4 5 5 ′0 4 5 3

No

No

No

No

Yes

Yes

No

No

Yes

No

Yes

Y¢S

Spectrometer：ShlmadzuAA・640－12atomicabsorptionandnameemissionspectrophoto・ m声ter・

l・amP ：Ho1lowcathodelamp（HamamatsuTV．） Flame ：Air－aCetylenename．

■ AdaptedfromJ．DeansandT．Rains2）．

－27－

Fig・1・CloseddigestionsystemforthedeterminatlOnOfleadandvolatileelemerLtS・

－28－

Resldts

Onegram（trlplicate，bottleNo・75）andtwogram（triplicate，bottleNo．100）samples， driedat85℃for4hoursinaJ”Ven，WeredigestedwithHNO3，＝F，and＝NO3inteflon beakersasdescribedinChapterIV－1・Blanksolutions（two）werepreparedinthesame manner・Thedigestedandblanksolutionsweremadeupto100mlwithdlSti11edwater．

TableI札

AnalyticalResultsforPepperbushbyAtomicAbsorptionSpectrometry

Element BottleNo．75 BottleNo．100 Average土Std．Dev．

1 2 3 1 2 3

K■（％）

Ca＊（％）

Mg蠣ら〟由

Mn＊ Zn Fe Na＊事

Rb Co Cu Ni Cd

L5

L3

7

41

10004124∩817つ‘

5 l

・1・3418342。1。79

つん0001つ▲10qノ′b48

・5ト3

1

7

4

つ‘10011qノ57つ▲OI

ト5ト34132113520975．23．12．8・66・6

31 1．32 1．33 1．31士0．01

4160士30 2100士20 340士7

205土7

101士1

75．0士2．6

23．7土0．4

12．1上0．3

8．72士0．43

6．67士0．05

6 1

4 331 333 1 204 198

9 101 102 3． 71．7 74，5

4．

m 1

7

Thevaluesarebasedonthematerialdriedat85℃for4hrsinanoven．

＊ 50timesdilutedwithO．1NHClO4．

■＊ AftersubtractionoftheNavaluefortheblanksolution．

イ．伽Je仰血αJわ〃qr用わ′肋〃－飽椚e

．右い′′血、．小ゴ（－甲J山〃敬一ビ（・〝川川廿甘

SamplePreparationandStandardSolutions

ForthedeteminationofPb，OnegTam（triplicateeachbottle，botdeNos．75andlOO）of PepperbushwastranSferred to anask arLddigestedwithIINO3and H202inthesystem ShowninFig，1．ThedigestionprocedurehasalsobeendescribedinChapterIV－2．

Thestandardstocksolution（1000ppmofPb）waspreparedbydlSSOIvingO．7994gofPb （NO3）2（99．9999％）inasmal1volumeofO．1MtJNO3anddilutingto500miwith0，1M HNO3・TheworkingsolutionswerepreparedbyappropriatedilutionofthelOOOppmstock SOlut10nWithO．1MHNO3．

Expe血entalConditions

Pbwasdeterminedbynon－flameatomicabsorptionspectrometrywithanIL－251atomic absorptionspeCtrOphotometerequlppedwithanIL・555atomizer・Backgroundcorrection

WaSPerfomeduslngadeuteriumhollowcathodelamp・TablelVshowstheexperimental COnditionsforPbdeteminatlOninPepperbush・

ー29－

Tat）1e Ⅳ．

ExperimentalCondltionsforDeterminationofPb

：1L－25latomicabsorptionspectrometerequlpPedwithIL－555atomizeL ：Pbhollowcathodelamp（HamamatsuTV．），De11terlumhollowcathode lamp（Westinghouse）forbackgroundcorrection． ：5mAforPbH．C．L．，25mAfordeuteriumlamp， ：283，3nm． ：1nm．

：75℃ror15sec．，100℃ねr15sec． ：400℃bTユOsec，，5SO℃払TユOsec． ：1900℃forlOsec．

Sp8CtrOmeter

Lmp

hmpCurrent

Analyticalune

Spect血Bandpa血

Dry Ash

Atomize

Re凱11ts

Table V．

PbContentorPepperbush．

neandyticalvduesareexpleS班dbasedonadryweiかtat85℃ねf4llrS．

BottleNo， SampleNo． Pbcontenthg／g）

75

100

Average土S．D．

エ・伽re〝乃血αJわ〃0／∬，Cb，∧b，月占，〃〃d

．ヾI・∫・■JナノりりJりふぃり、1卜lゾ′・申い亘■

SamplePreparationandStandardSolutions

・The dlgeStion ofpepperbush sampleswasperfomedwithHNO3，HFandHClO4aS describedinChapterlV－1・One伊江mS卸Ipleswe托心野Stedandd血tedtol00ml・Fo一也e deteminatioJ10fKandCa，thesolutionswerediluted50timeswith0．1MHC104．Tothe standardworkingsolutionsofNaandRb，KwasaddedtoglVe150ppmofKwhichcorres－ pondedtotheKlevelinthesamplesolutions・CawasaddedtoeachoftheworklngSOlutions ofSrtoprovidethebalconcentrationof150ppmCa・

－30－

ExperimemhlConditions

TableⅥ．

ExperimentalCondltionsfortheDeterminationof K，Ca，Na，RbandSrbyFlameEmissionSpectrometry

h

鋸Wi。t曲

Flow Rate Air・Acetylene

（1／min）

AnalyticalLine （nm）

Elem（！nt

766．5

422．7

589．0

780．0

460．7

qノ q／ Qノ qノ 9

5 5 3 3一斗 つム ▲ソ】 へ∠「－つ］

0 0 0 0 0

aNa

KC馳Sr

Spectrometer：ShimadzuAA－640－12atomicabsorptionandnameemisslOnSPeCtrOPhoto－

meter．

Flame ：Air・aCetylenename．

Results

Tabl（さ ⅥⅠ．

AnalyticalResultsforPepperbushbyFlameEmissionSpectrometry

Bottle 75 Bottle 100 Average±S・D・

Element 1 2 2 3

K（％）

Ca（％）

Naレg／g）

Rb

Sr

54朋066・2‖

． l l 1 7 つ】

3

5 ．．

3

54

．一2

3

5 ．．

3

1．53虹）．01

1．32土0．04

106土3

74，7士2．6

30，1土1．6

4 5

029・0

．ぷ‖ 1 1 1 7 2

2

～ ．．

3

Theanalyticalvaluesareexpressedbasedonadryweightat85℃for4hrs・

－3l－

References

l・Rains，T．C．（1977）：AtomicabsorptlOnandflameemissionspectrometry．A．Detemi・ natlOnOfbery11ium）Cadmium，COPper，magneSium，manganeSe，merCury，nickelandzinc by atomic absorption，and ofcalcium，POtaSSium，rubidlum and sodlum by蝕Ⅲle emisslOnSpeCtrOmetryinbiolo顔Calandbotamicalmaterials．NBSSbecia］hJbticatTOn 492：49■65．

2・Deans，J・A・＆Rains，T．C・（1971）：StaEldardsolutjonsforL］amespectrometry．In： f7ameemisstonandatomicabsorptionspectromeり・PeanS，J．A．＆Reins，T．Cリed．），2， Chapter13，MarcelDekker，NewYork，

Rains，T，C・＆Menis，0．（1976）＝Anintercomparisonofflameandnonflamesystemsin atoTnicabsorptioIISPeCtrOmetry．JⅥげ5＞ec由JhLbLkatTOn422，VoJ．2：1045．105l．

JRains，T・C・（1974）：Atomicabsorptionspectrometry－generalconsiderationforthe applicationofexperimentaltechniques．勘ecialTbchnica］hJblLca（ion564，AmericaJI SocietyforTestingandMaterials，p．50－‘7．

Rains，T．C・（1969）：Chemicalaspectsofatomicabsorption．S＞echltteChnicalpubtica－ （Lon443，AmericanSocietyforTestingandMaterials，p，19－36．

－32一

CHAPTER V

ELEMENTALANALYSISOFPEPPERBUSHBYICP

EMISSIONSPECTROMETRY■

C．W．M．McLeod，N．FurutaandM．Nishikawa

‘血gr（X7〟CJわ〝

Thestandardreferencematerial（SRM）certifiedforelementalcompositionisindispensa－ bletoanalyticalme血odsifaccuracyandprecisionaretobeachleVedandmaintained・To meettheever・increaslngdemandfromthemanyareasofscienceandindustry，gOVemment－ supportedbodiesinanumberofcountriesareactivelyinvoIvedin仙eissuanceofvarious categoriesofSRM，s．1nJapan，theNationalInstituteforEnvironmentalStudies（NIES） recentlyinstigatedanSRMprogrammetoservetheneedsofenYironmentalscientistsandthe botamicalrefeTenCelnaterlal，Pepperbu血，isthenrststandardtobeissued・Areportonthe preparationofPepperbudlandcomparisonofelementalcontentwlthotheTbotaruCalSRM’s hasbeenpubllShedl）．0thprcategorleSOfenvlrOnmentalSRM’salreadypreparedinclude PondSedimentandChloreuaandanalysisisunderway，

Thecertincationprocess，inthenrstinstance，requlreSanalyticaldatafortheelementsto beobtainedflomindependentandestablishedanalyticaltechniques・Inductivelycoupled plasma（ICP）emission sp占ctrometry，a relatiYely newtechnique，hasmadeasignincant impactintheelementalanalysis鮎1d2）anditisconsideredthatitcanmakeanimportant contributiontotheSRM certificationprocess・NoteworthyfeaturesoftheICPtechnique havebeendlSCuSSedintheliterature2，3）andincludetheapplicabilitytometalsandnon－ metals，ahighsensitivityforal1elementswithaslnglesetofplasmaoperatingcondltions，a simultaneousmultielementcapability，awidedynamicrangeandarelativefreedomfrom interelementmatrixinterference．Thepr血ary objectiveinthecertincat10nprOCeSSisthe attainmentofaccuracy且ndfromthispointofviewtherelativeabsenceofmatrixinter－ ferenceisaparticuladyslgnlnCantfeature・

Inthisreport，adescriptionoftheplasmaemissiontechniqueandinstrumentationatan elemeTltarylevelisfirstpresentedandthenconsiderationlSglVentOtheselectionofplasma operatingcondltions forsimultaneousmultielementanalysis（SMA）・ThevarlOuSfactors whichmayinfluencetheaccuracyandprecisionoflCPmeasurementaredlSCuSSed・The analyticalmethodisdescribedindetai1andthereliabilityoftheresultsforPepperbushis assessedbyreferTingtothecorrespondingdataforOrchardLeavesSRM（NadonalBuIeauOf Standards，U．S．A）．

＊ PresentedinpartatJointUS－JapanSymposiumonStandardReferenceMaterials，Kori－ yama，Japan．

一33－

飽馴最知叫血肌肌用勅W酬棚肋

Theinductively coupled argonplasrna（1CP），ahightemperatureexcitationsource，is fomedbythecouphgofionizedargoIIWlthapowerfulradiofrequency（RF）鮎1d．When Sample，uSuallyin1iquidform，isintroducedtotheplasma，aSanaerOSOlmistviapneumatic nebullZation，theaeIOSOlparticlesundergovariousprocessessuchasdesoIYation，decomposi・ tion，atOmization／excitation andionization／excitation．The atomic andionic emission Characteristicofanalyteelementsisreceivedbyaspectrometerandthemultielementslgnals arepfOCeSSedbyadedlCatedcomputer．

Theexpe血Ientalarrangement forplasmapIOductionconsists，typically，Ofa3－Wal1ed qua∫tZtube coupledtoaspIay Chamberand nebulizer，AcopperiJlductioncoil，through dlich codant Wateris passed，SurrOuIldsthe top endofthequartztube・Aschematic diagram ofaconventionalsystemis騨VeninFig・1■TwomalnargOngaSflows，namely COOlant（outemlOSt～151／min）andnebulizer（innemostflow－11／min）servctosustainthe Plasma（andpTeVentOVeheatlngOfthetorch）andtotransportthesampleaerosoltothe Plasma・UseoftheauxiliaryaTgOngaSflowisoptional・ForimitiatlOnOftheplasma，ateSla CO止isusedtoionizetheargongasstreamandprovidea“s声ed”ofelectronswhichbecomc

thermany excitedinthealtemating RF neld．ArapiddseinelectronenefgyPrOmOteS furtherionization ofthe argonandthecouisionalexcitalユOnPrOCeSSeSthatensuedevelop andmaintalntheflame－1ikedischarge，ThesampleaerosolgasstreampenetrateSthecentreof the plasmaand■passesupthroughanarrow centralaxialchannel．Thehightcmperaturcs WhichtheanalytcexpeJiencesintheinertargonabosphereandtherelativelylonganalyte resldence times TendeTthelCP an exceptionalexcitation source for analytlCalemission SPeCtrOmetZy・

lnth1SStudy，tWOindependentICPinstrumentsareutilized，aCOnVent10naldirectreader andaprogrammablemonochromatorsystemincorporatingasiliconintensifiedtarget（SIT） detector・Abriefdescription ofequlPment andoperatingconditionsforbothsystemsare giveninTableI．Thedirectreadersystemisacommerciauyavanableinstrument（Jarrell－Ash Atomcomp，Mode1975）andhasthe capabilityofthe simultaneousdetermination of26 elementsatsensitivity1eYelswhlCharetypicaJJyatthepartperbiuionorder．Aschematic diagramofthemaincomponentsoftheinstrumentisgiveninFig・2・Thepolychromator， O．75mPaschen－Rungeunit，COntalnSpr¢al1gnedphotomultlPliertube（PMT）detectoISand respectlVeeXltSlitssituatedalongthefocalcurveatappropriatewavelengthpositions・The Opti叫con免guTationis essentially触edbutextendingelementcapat血ityis．possibleby instalIingadditionalPMT’satthedesiredwavelengdlpOSitions．Theemissionwavelengthsfor elementsprogIammedintothe system areglVenlater．TheinstrumentatlOnalsofねturesa Single channelmonochromator to provide forvarlable wavelengthmonltOrlngandwaYe・ 1eng仙scammg．

Sy5tem OperatlOn，undercomputer control，is perfonned via thehlgh．speedteletype device，1nputcommandsareenteredasaseriesoflettersinordertoperforrnstandardisation andto血plementtheanalysissequence・Themultielementemissionsignalsafterdetection andprocessingaredisplayedontheteletypeusuauyinunitsofconcentration・

Novelresearchinstfumentationincorporating a siliconintensified target（SIT）asthe primaryradiationdetectorhasrecentlybeendeveloped4），Thecomputer・COrLtrO11edsystem employs血eslew・SCanteChni甲eWheTe坤themonochromatoTispTOgrmmedtoaddress，血 SequenCe，0nlythe desiredemiss10n WユVelengths（wavelengdlreg10nS Ofnointerestare rapidly bypassed）to provide a rapid岳equentialmultlelement analysis capability・The expe血erLtalsystemisdepictedinFig・3・Analysisproceduresthroughappropriatesoftware

ー34－

Ptasma Obs．Ht．

15－20mm abovecoil） （

ヽ川■ 一叫 lnduction Coil

Torch Diam．

20nl…

R．F．R）Wer くq．7－2．0仰〟 2了．12MHz）

Co0lant Ar（181∧Tlin）

lgniti6nCoiJ ▼Auxiliary

Ar （OPtionaり

】■ISpray Chamber

saL、Ple（。Ptakerate，0．3－2．Oml／min）

Fig・1・ICPtorchassemblyandcrossflownebulizer・

－35－

areagaincontronedentirelyfromtheteletype・ The StT detectoTisone ofanumbeTOfsdlidstatemultichanneldevicesthathasbeeJ】

血dingincreasinguseinspectroscopicmeasuremcntsystems5），Thedetectorisplacedatthe exitportofthemonochromator（exltSlitremoved）andprovidesasirrlultaAeOuSmOnitorlng CaPabilityoveraspeci伍cwaYelengthrangewhichisdeterrninedprimari1ybythereciprocal 1ineardispersionofthemonochromatol，FoTtheptesentsystema5LmSPeCtraZwlndow， ＄ubdlVidedinto500electronicchannels，isdisplayedacrossthetubeface．TheSITconsists OfanarrayofllghtsensltiYeelementswhichdevelopachangepattemonexposuretouv／vis radiation・Thechangepatte－nisaddIeSSedbyascanningelectTOnbeamandthTOuglthe Op出ca】multicム肌ne】皿dy5er（OMA）controIuI出血esj卯山c弧bedゆIayedinrealt血eon anoscnloscopeorstoredinmemoryforfurtherprocesslng，e・g・SigrLalaccumulat10n・The OMAunithastwoseparatememoryareas（memoryAandmemoryB）forsignalstorageso that plasma backgIOund・COrreCted spectra（A・B mode）caJlreadny be affoJded．Such emiss10nSPeCtraareOfpar臼Cularvalueforthestudy，Classlficationandcorrectionofplasma interference effects anditisinthis respectthat SITdetectlOnofferscertain adYantageS relativetoPMTdetectionforplasmaappucations・ThequantumefRciencyoftheSITis relativelypoorindlelowuvbutforanalyteemissionwavelengthsgreaterthanaPPrOX皿ate－ 1y250nm，detectionl血itsforSITandPPTdetectionarecomparable6）．

ForPepperbushanalysisbothinstrumentsarcusedtoprovidequantitativedatabutin additionforqtlalitativestudiessuchascheckingforpotential函smainterferenceeffects，the SITinstrumentisemployed・FurtherdetailsregardingtheperLbrmance chaT8Cteristjcsof bothinstrumentSaTeindicatedinlatersections．

Tablel

De5CdptlO∫10f血5tru汀－e地点o乃andOper且血gCo‡通日0れS

Jarrell・AshAtomcomp PurposeBuilt （Compuf釘・C珊‡一曲ed）

Polychromator

DisperslOn，0．53rlm／mm

hlstorder．

PMT（Multic血相el）

nasmaThem

27

4 tums

Spectrometer Monoch【OmatOr（Jobin YYOn） Dispersion，0．4nm／mm

inlstorder．

SIT（Singlechannel）

Shimadzu

27

2 tums

Detection

GenelatOr

FlequenCy（MHz） badCoil

NormalOperatingConditlOnS FoⅣ征dPoweT（Kw） Cool∽f仰mjn） Ca血eT（りmin）

Auxiliary（1／min）

． J

8

1 1⊥ l▲ （U

．60 6

ObservationHeight（mm） 19 17

Nebulizer and Cross－nOWtype Concentric type

UptakeRate（mi／min） ～1・3 ～2・5

ー36－

l● SAMPL【

Fjg・2・M血componeれ捷Of血ectrea血glCP餌血5im5匹CtrOmeter．

Fig・3・MaincomponentsofSIT・basedICPemiss10nSPeCtrOmeter・

ー37－

加叫漉励払血如叫刑

1．htroduetoけRemaTks

Inthis sectlon，features and operationalcharacteristics oftheICP technique are introducedandattentionisdrawntovarioysfhctorswhichmaycontrlbutetoaccuracyand precisionofICPmeasurement，Ofconcemarefactorssuchassampledissolution，．StarLdard SOlution composition and callbTation，Physicaland spectralinterference andinstrument Stability・At nrst，the principalplasrpa operatlngparaJneterSareCOnSideredsincethey detemlinetoagreatextentanalyticalperEormancechaTaCteristics・

2．PrhcipalPlasmaOpera血gConditioI漬

Fors血ultancousmultielementanalysis（SMA）abasicrequirementforal1techniquesis 山eselectionofa血如setofope∫血gpar抑e血；jM－叩Cおe5C叩rOjざSa托made betweensensitivity，interferenceandapplicability・FortunatelyinplasmaemlSSlOnSPeCtrO－ metryasinglesetofoperatm％COnditionscanbefoundto由VehlghsensitivityfoTmany elements and this contrasts with other techniques such as flame spectrometrywhere opthumconditionsforelementsarevelydependentonnameconditions4，7）．

InlCP emission spectrometJy3plasmaopelatJJ）gPa（NneteTS，namelyrad10丘equency （RF）p？Wer，Carriergasnowrateandtheplasmaobservationzone・havebeenfoundtoplay adecisIVerOleindetemllningdetectionperLbmlanCeforaparticularelemerLt8）．Toobtain 血e optimum plasma operating conditions for a particdar element one approachis to maximizethesignaltobackgroundratio（S／B）fortheelement，itbeingassumedthatthe majumumS／Bcorrespondstothemaximumsigrlaltonoiseratio（S／N）andhencethelowest detectionlimit．Thisapproachisvalld forthc．sesystemswhosenoiseis5011rCe－dominated andwherethestandarddeviation ofthebackgroundnoiseisdirectlyproportionaltothe backgroundleve19，10，11）．肘ore血eTeS山sfo一山eS／Bop血kation血曲Sare叩駅nted， SOme basIC COmmerLtS COnCerrLingthe role ofthe principaloperating parameters are necessary・Firstletusconsidertheuv／visspectrumOftheplasmawhichis血owninFig．4． The spectrumiscomplexwithmanyArlinessuperimposedonthecontinuumbackground and spectralfeaturescharacteristic ofspecies such asH，OH，NH，NO，CandCNcanbe identined・The r碩On丘om200－350nmispartlCularlysuitableforanalysissirLCePlasma backgrdundisrelativelylow andalsobecauseintense atomicandionlC emissionlinesof elementsarelocatedin血isTegion（seeTablelI）．

Atpositionsneartotheloadcoil（observatlOnregionapprox．0・10rnmabovethecoil）， dle plasmatemperatureishigh（7000・8000K）andtheargoncontinuumbackgroundis intense，ThlSredonisnotusuallyfavourableforobsefVlnganalyteemisslOnOnaCCOuntnOt Onlyofthehlghback騨OuIldbutalsobecausetherelatlVelysho［tanalytet8Sidencetimeis insufncienttovaporizeandexcitetheanalyte．Onmovinguptheplasmathetemperature graduallydecreasesandobservationzoneshighintheplasma，e．g．30－60mmaTealsonot ㌣SuallyfavourablewheretemperaturesapproachiJlgthatofchemicalcombustionnamesare obtained．Anintermediatere辞On，15－20mm，Whichcorrespohdstoanalyteresidencetin1eS ofthemillisecondorderisfavourablefofanalyticalmeasur¢ment．

1ncreaseof血eRFenergyresultsinarisehplasmatemperatureandacorrespondingnse

intheplasmabackground（theplasmavolumeandluminosityarealsoincreased）・Although for most el印IerltS maXmum emisslOnintensityis favoured by ahightemperature，the

magnitude ofthebackgroundintensityleveldeterminestoagreatextenttheopt血um

ー3β－

300 400

WJNELENGTH（nm）

Fig・4・W・ⅤISspectrumora柑OnICP plasmaoperatugconditions，R・F・POWerl・5kw；Observationheight・11mm；SamPle uptakerat¢～1．Oml／min・

powerlevel． The3rdirnportantvariable，theargoncarrlergaSnOWrate，SerVeStOnebulizethesample

andtotransporttheaerosoldropletstotheplasma・Forsumcientlyhighcarriergasflow rates，the姐mplepenetratestheplasmaandtravelsupthenarrowcentralaxialchannel．At

Veryhlghflowratesperformanceis，however，impairedduetocoolirlgOftheplasmaandalso

Table tl

Op血umValuesofPrincipalICPOperatlngParameterSandDetectionL如Iits

Power Obser．Ht Sample Uptake DL．＊

Waveleng血 above coil Rate

（nm） （Kw） （mm） （mi／min） b）Pm）

Bement

32臥1 1．1

308．2 1．1

193．7 0．9

249．8 0．9－1，0

313．0（ⅠⅠ） 1．7

317．9（Ⅰり 1．1－1．2

22臥2＊事 1．0．1．1

22乱6（11） 1．1

267．7（Ⅰり 1．2・1．3

324．8 0．95

259．9（Ⅰり 0．9－1．0

253．6 1．1

766．5 0．85

279．6（1Ⅰ） 1，1

257．6（叫 1．6

202．0（Il） 1．3

589．0 0．8■．9

231．6＊＊（Il） 0．85

214．9＊＊ 1．2

（ⅠⅠ） 5

196．0 1．0

288，2 1．1

24 1．1

20－22 1．0－1．1

18．20 0．9．1．0

19－21 1，1

16．18 0．95

0．001

0．017

0．020

0．001

0．000き

Ag刃ASB鮎CaCdC。CrCuFeHgKMgMnM。NaNIPPbSbSeSiSnTiVZn

1，0－1．1 0．005

0．9．1．0 0．001

0．9－1．0 0．002

0 ▲U

－㌧ 1㌧

Qノ 8 8 0 つ‘ l l l つ】 つん

0．005

0．0008

0．002

0．014

0．008

0．00007

0．0006

0．005

0．0008

0．006

0．04

0．011

0．00（i

O．022

0．010

23 1．2

16．18 1．1

12 ＞1．6

17 0．95

17－柑 1．0

18－19 1．0－1．1

14－15 1．1・1．4

18 0．9．1．1

16．18 1．2

19 1．1

19－20 1．1

17．18 1．0．1．1

21 1．0－1．1

18丸9（Il）

334．9（ⅠⅠ）

292．4（ⅠⅠ）

213．9

18 0．85－0．95 0．021

19・20 0．95 0．0008

20 1．1 0．003

16・18 0．9 0．002

1．1－1．2

1．0

1．1

1．l

＊ PlasmaoperatingcondltiollS：POWer，l・1Kw；Observationheight，19mm；flowrate，

1．Oml／min． （Il）referstoiomicliJle． ＊● refersto2ndorder， 1datawereobtainedbymonochromatordetection．

－40一

becauseofareducedanalyteresldetlCetin1e・PooraJlalytlCalperfc．rmanceisalsoobtainedat lowcarriergasnowrateswheretheaerosoldropletvelocityisnotsumcienttopenetratethe plasmaandinsteadtheparticlespassthroughtheouterregiorlSOftheplasma2），It血ouldbe mentionedthatnebulizationemciencyisabout5％．

TodeteminetheoptnlumOPeratingconditionsforsimultaneousmultlelementdetermi－ nations fhe maxin1um S／BforelementsprogrammedintothedlreCtreaderwasobtained， Experlmentauy，thisinvoIvedlocation ofthemaximum S／B fortheindivid11alelements when one plasma parameter was varied whilethe other two were nxed（e．g．stepwise increm？ntSOfpowerforfixedobservationheightandsampleuptakerate）・lnthisway，it wasposslbletoobtainasetofplasmaoperat山一gCOnditionswhichyieldedthemaximumS／B fortheeachelement．Graphicalpresentationofthesignal（S）andbackground（B）behaviour for4elements（Na，Cu・Zn，Ca）asafunctlOnOftheplasmaoperatingconditionsSgivenin Fig．5（1，2，3），TheekmeTltSNa，Cu，ZTlandCawerechosellfor山ustratlOrISlnCethejr emissionbehaviourwasconslderedtoencompassthat oftheelementsinvestigatedinthis Study（seeTableII）．

Forthe power variation study（Fig．5．1），element emissionintensityincreaseswith increasingpower（not cont血10uSly up to2・Okw fortheatomlinesofNaandCuwhere levening－Offoccurs）aJldmamainthqrespectiveScurvesdonotoccur・Theaccompanyやg ICreaSeOfplasmabackground（withpower）atal1wavelengthscounteIaCtStheemisslOn mcreaseandtheoptimumS／Btendstooccuratlowpowerlevelsforthe4elements（l，1 kw）．ItshouldbenotedthatthebehaviouroftheZnatomiclineissimi1aTtOthatoftheCa ionicIjLteandnottotheCuatomicline，de丘nltem批iIllaOCCurriJlgfortheS／Bcurvesofthe fomertwoelements．Thisisinagreementwiththe鮎dlngSOfBemanandMcLBren8）who COnSlderedthattherelatlVelyhlghexcitationenergyoftheZnatomline（5．8eVcf．3．8eV forCu）isresponsibleforemiss10nbehavlOurSimilartothatofioniclines．Thebehaviourof theotherelementsinvestigated（seeTablelI）utilizingeitherionlCOratOmicemissionwas slmilartothatofCa．

hexplainingtheemissiondependenceonplasmaobservationheight（seeFig．5．2），the important fhctors to considerare（l）theprogressivedecreaseinplasThatemperaturewith lCreaSingobservationheightand（2）theanalyteresidencetuneappropriatetothemeasur一

皿gPOSlt10n．Na（alsoK）beinganeasilyionizedelementfaYO11TSalowobservationheight COrreSpOndingtoashortplasmaresidencetimetoprovidethemaximumatomicemission・ HlgherobservationheightsarenecessaryinthecaseofCu，Zn，CatonSureCOmpletevapori－ Zation／excitat10nandincontrasttothatforNawell－prOnOunCedmaxlmaCentredaroundthe 15mmregionwere obtalnedfromthe respectiveScurves．The continuumplasmaback－ grounddecreaseswithincreasingobservationheightsothatthema氾mumintheS／Bcurves are dllftedtosllghtlyhigherheightsrelative tothat fortheScurves．Znisanexception wherebecauseofthepresenceofNO，thebackgroundcommencestoincreasewithincreas－ ing observation height beyondabout17mm・Thatis，the NO populationinFreaSeS progressivelywithobserYationheightbeyondapproximately17mmduetoairmtralnment and consequently the background emission at213．9nm alsoincreases．This effectis responsibleforthecoincidenceoftheZnSandS／Bcurves（Cf．Cu，Ca）．

lnde血Iiteincreaseofthesampleup